Access to and use of human specimens is an essential part of the cancer research and drug discovery infrastructure, enabling researchers to, inter alia, correlate gene mutations/polymorphisms with particular cancers, identify drug targets, develop therapeutic compounds, guide therapy decisions, and understand drug metabolism. Research using human specimens can help predict drug response and toxicity, and clinical outcome. Many different types of biological specimens are required to support these studies: normal and malignant tissues, blood, blood products, other bodily fluids, as well as proteins and nucleic acids that can be extracted from them.
Formalin-fixed, paraffin-embedded (FFPE) tissue, which is one common method of preserving specimens, is a frequent source of biological specimens. Formaldehyde fixation, however, causes cross-linking between nucleic acids and proteins, the reversal of which leads to fragmentation of DNA and RNA. And, the paraffin-embedded tissue sections require de-waxing to allow penetration by aqueous solutions prior to analysis of nucleic acids.
For example, in one extraction procedure a razor blade is used to scrape FFPE sections, which are then transferred into microfuge tubes for processing. The traditional method of paraffin removal involves organic extractions using xylene and graded alcohols. This procedure is time-consuming, cumbersome, and requires special handling, as xylene is a highly toxic chemical that emits noxious fumes.
Phase extraction de-waxing protocols are time consuming and laborious. And, the repeated handling, aspirations and tube transfers can result in non-quantitative harvests of the nucleic acids. Moreover, repeated vortexing of the sample and exposure to harsh solvents can cause additional sample degradation. Commercial kits are available that optimize the nucleic acid extraction process but the resulting quality and quantity of nucleic acid recovered from FFPE tissues is variable.
Problems exist in the quantitation of nucleic acids from preserved clinical specimens such as FFPE tissues. Extracted nucleic acid quality and quantity is often affected by both sample collection and extraction procedures due to degradation and fragmentation. This can compromise, for example, the ability to measure the extracted nucleic acids. Qualitative and quantitative assay errors often result when these extracts are evaluated by standard analytical techniques. Moreover, incomplete extractions can introduce error into calculations, such as mRNA copy number determinations.
A variety of methods exist for attempting to assess the quantity and quality of extracted nucleic acids. For example, 260 nm/280 nm absorbance by spectrophotometry can assess high molecular weight (intact) genomic DNA. But nucleic acid fragmentation can result in highly erroneous results and an overestimation of nucleic acid amounts. The use of intercalating dyes is also widely used, however, the accuracy of assays based on these dyes are significantly impacted by an over-abundance of small nucleic acid fragments in degraded sample.
Sensitivity of detection of a genetic variant is dependent upon the quantity and quality of nucleic acid available for analysis. Although existing methods that are used to determine nucleic acid quantity and quality are useful in limited circumstances, they generally involve additional testing prior to genetic analysis of a sample. A simple method to evaluate the quantity and quality of DNA or RNA simultaneously with the genetic analysis is therefore needed.